METHOD FOR PREPARING GLUCOSE FROM STARCH SUGAR BY USING ACIDOTHERMUS SP.-DERIVED DEBRANCHING ENZYME HAVING HEAT RESISTANCE AND ACID RESISTANCE, AND GLUCOSE PREPARED THEREBY

Abstract

The present application relates to improving the production yield of glucose to be prepared by using starch sugar as a raw material and using an Acidothermus sp. derived enzyme. The present application can replace an acid saccharification method, which produces a bitter taste through a hydrolysis reverse reaction, and, compared to a enzymatic saccharification method in which a conventional debranching enzyme is used, has an advantage of enabling costs to be reduced since a relatively high yield can be ensured by reducing side reactions of a saccharification reaction.

Claims

1. A method for producing glucose from starch sugars, comprising: treating a substrate solution containing starch sugars with a debranching enzyme derived from a heat-resistant and acid-tolerant Acidothermus sp. and a diastatic enzyme.

2. The method according to claim 1, wherein the debranching enzyme derived from an Acidothermus sp. is produced by inserting a gene encoding the debranching enzyme into a vector to produce an expression vector, transforming the expression vector into cells, and mass-producing the transformed cells.

3. The method according to claim 2, wherein the vector is pET-28a(+).

4. The method according to claim 1, wherein the debranching enzyme derived from an Acidothermus sp. is an isoamylase.

5. The method according to claim 1, wherein the Acidothermus sp. is Acidothermus cellulolyticus.

6. The method according to claim 1, wherein the transformed cells are any one of E. coli, a microorganism belonging to the genus Bacillus, yeast, a microorganism belonging to the genus Corynebacterium and a plant.

7. The method according to claim 1, wherein the starch sugars are amylopectin-type starch.

8. The method according to claim 1, wherein the starch sugars are liquefied ones.

9. The method according to claim 1, wherein the starch sugars are derived from any one crop selected from corn, tapioca, potato, and rice.

10. The method according to claim 1, wherein the substrate solution containing starch sugars is treated with the debranching enzyme derived from an Acidothermus sp. and the diastatic enzyme at a pH of 2 to 6.

11. The method according to claim 1, wherein the substrate solution containing starch sugars is treated with the debranching enzyme derived from an Acidothermus sp. and the diastatic enzyme at a temperature of 40 C. to 70 C.

12. The method according to claim 1, wherein the total unit ratio of the debranching enzyme derived from an Acidothermus sp. to the diastatic enzyme is from 2:1 to 60:1.

13. A method of using a debranching enzyme derived from a heat-resistant and acid-tolerant Acidothermus sp. in the production of glucose from starch sugars.

14. A debranching enzyme derived from Acidothermus cellulolyticus that is active at a temperature of 40 C. to 70 C. and a pH of 2 to 6.

Description

DESCRIPTION OF DRAWINGS

[0030] FIG. 1 shows several hydrolases and pathways for the degradation of starch sugar.

[0031] FIG. 2 shows several debranching enzymes and pathways for the degradation of starch sugar.

MODE FOR INVENTION

[0032] The present invention will be described in more detail with reference to the following examples. These examples are merely illustrative to assist in understanding the invention and should not be construed as limiting the scope of the invention.

EXAMPLE 1

Gene Isolation and Recombinant Protein Production

[0033] 1) Production of Recombinant E. coli

[0034] Forward and reverse primer sequences including portions of both end sequences of a debranching enzyme gene derived from a heat-resistant and acid-tolerant Acidothermus cellulolyticus and the recognition sequences of restriction enzymes NdeI and HindIII, respectively, were used for PCR.

TABLE-US-00001 (Forward)5-GAA-TTC-ATG-CCG-GAA-3 (Reverse)5-GAA-TTC-TTA-GAG-GAC-3

[0035] As a result, a PCR product of 2.1 kb was obtained. The acquired DNA fragment was inserted into pET-28a(+) vector and transfected into E. coli BL21(DE3). The DNA has the sequence set forth in SEQ ID NO. 1. The transformed E. coli was spread on the plate media containing kanamycin. Kanamycin-resistant strains were primarily screened and separately cultured in liquid media. DNA of Kanamycin-resistant strain was purified and digested with both NdeI and HindIII. The finally selected strain was found to have 2.1 kb and 5.3 kb DNA fragments. The sequence was analyzed using an automatic sequencer. As a result, the sequence was found to be identical to the gene sequence of the desired debranching enzyme.

[0036] 2) Recombinant Protein Production

[0037] A debranching enzyme (isoamylase) was derived from acid-tolerant and heat-resistant Acidothermus cellulolyticus by the following procedure.

[0038] A test tube containing 5 ml of LB liquid medium was inoculated with frozen-stored recombinant E. coli strain BL21(DE3). The inoculum was cultured in an incubator at 37 C. until an absorbance of 2.0 at 600 nm was reached. The culture broth was added to 500 ml of LB liquid medium in a 2 L flask, followed by main culture. When the absorbance of the culture at 600 nm reached 0.4, 0.1 mM isopropyl -D-1-thiogalactopyranoside (IPTG) was added to induce mass expression of a debranching enzyme. The temperature was maintained at 37 C. with stirring at 180 rpm during culture. Even after the addition of the IPTG, the culture was continued at 20 C. with stirring at 120 rpm. The culture broth of the transformed strain was centrifuged at 6,000g and 4 C. for 20 min and washed twice with 50 mM Tris-Cl buffer. Then, Ni-NTA binding buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0) was added and the cell solution was disrupted using an ultrasonic homogenizer. The cell lysate was centrifuged at 13,000g and 4 C. for 20 min. The supernatant was collected as an enzyme extract and purified using a Ni-NTA superflow column to accurately characterize the enzyme. The purified enzyme was found to have a molecular weight of about-75 kDA, as determined by SDS-PAGE.

EXAMPLE 2

The Measurement of An-Enzymatic Activity by HPLC

[0039] 1) The Activity of the Debranching Enzyme (Isoamylase) Derived from the Acid-Tolerant and Heat-Resistant Microorganism (Acidothermus cellulolyticus) was Measured by HPLC under the Following Conditions.

[0040] HPLC analysis conditions: [0041] Detector: RID [0042] Flow rate: 0.6 ml/min [0043] Sample injection vol.: 10 l [0044] Column: Bio-Rad HPX 87C [0045] Solvent: D.I. Water

[0046] The total time for analysis was set to 20 min.

[0047] The activity of the debranching enzyme (isoamylase) derived from the acid-tolerant and heat-resistant microorganism (Acidothermus cellulolyticus) was confirmed by the production of glucose from corn starch as a raw material through multiple enzymatic reactions with a diastatic enzyme (Glucoamylase). The enzymatic reaction conditions were as follows.

[0048] First, a diastatic enzyme (Glucoamylase, DuPont company) and the debranching enzyme (isoamylase, 50 mM sodium acetate buffer, pH 4.3) produced in Example 1 were used in a total unit ratio of 1:4. Multiple enzymatic reactions with liquefied starch having a dextrose equivalent (DE) of 9.5 were performed at a temperature of 60 C. for 72 h. The reactions were quenched by heating at 100 C. for 5 min. Then, the amount of glucose produced was measured by HPLC. The results of HPLC analysis revealed that the yield of glucose from the corn starch was 96.2%. The glucose yield was calculated from the ratio between the areas of peaks corresponding to the enzymatic reaction products.

EXAMPLE 3

The comparison of yields of glucose produced using the debranching enzyme (isoamylase) derived from Acidothermus sp. and commercial debranching enzyme (Pullulanase)

[0049] The yields of glucose from starch using the debranching enzyme derived from Acidothermus cellulolyticus and a commercial debranching enzyme (Pullulanase, Novozyme company) were investigated. The enzymatic reaction conditions were as follows.

[0050] First, a diastatic enzyme (Glucoamylase, DuPont company) and each of the debranching enzymes (isoamylase and pullulanase) were used in a total unit ratio of 1:4. Multiple enzymatic reactions with liquefied starch (DE 9.5) were performed at a temperature of 60 C. for 72 h. The reactions were quenched by heating at 100 C. for 5 min. Then, the amounts of glucose produced were measured from the ratios between the areas of peaks corresponding to the enzymatic reaction products by HPLC. As a result, the reactions using the debranching enzyme (isoamylase) derived from Acidothermus cellulolyticus gave a higher glucose yield than the reactions using a commercial debranching enzyme (Pullulanase). Starch sugars, including di-(DP2) and higher saccharides, were analyzed. As a result, when the debranching enzyme (isoamylase) derived from Acidothermus cellulolyticus was used, the proportions of the unreacted products (DP3, DP4+) were reduced, indicating relatively high yield of glucose.

TABLE-US-00002 TABLE 1 Diastatic enzyme Debranching enzyme Glucose (%) DP2 (%) DP3 (%) DP4+ (%) Glucoamylase Isoamylase derived from 96.20 2.57 0.39 0.84 (DuPont company) Acidothermus sp. (Ratio 4) (Ratio 1) Pullulanase (Novozym 96.10 2.16 0.64 1.10 company)(Ratio 4)

[0051] As shown in Table 1, the use of the isoamylase derived from Acidothermus cellulolyticus in the present application led to a higher glucose yield due to its higher activity than the use of the pullulanase known to be optimal for mass production. The proportions of the unreacted products (DP3 and DP4+) with relatively high degrees of polymerization were smaller when the said isoamylase was used than when the pullulanase was used. From these results, it can be concluded that the application of the inventive isoamylase to enzymatic reactions leads to an increase in glucose yield.

EXAMPLE 4

The Changes in Glucose Yield with varying Concentrations of the Debranching Enzyme (Isoamylase) Derived from Acidothermus sp.

[0052] The changes in glucose yield with varying concentrations of the debranching enzyme (isoamylase) derived from Acidothermus cellulolyticus were investigated. The enzymatic reaction conditions were as follows. Multiple enzymatic reactions with liquefied starch (DE 9.5) were performed at a temperature of 60 C. for 72 h, wherein a diastatic enzyme (Glucoamylase, DuPont company) and the debranching enzyme were used in total unit ratios of 1:2, 1:10, 1:20, and 1:60, respectively. The reactions were quenched by heating at 100 C. for 5 min. Then, the amounts of glucose produced were measured from the ratios between the areas of peaks corresponding to the enzymatic reaction products by HPLC. As a result, the higher the ratio of the debranching enzyme (isoamylase) to the diastatic enzyme, the higher the glucose yield. These results appear to be because the higher activity of the debranching enzyme (isoamylase) derived from Acidothermus cellulolyticus leads to the production of smaller amounts of by-products (DP2) formed in the starch hydrolysis and the degradation of larger amounts of the unreacted products (DP4) by the diastatic enzyme, resulting in an increase in glucose yield.

TABLE-US-00003 TABLE 2 Diastatic enzyme Debranching enzyme (glucoamylase, DuPont (Isoamylase from company) Acidothermus) Glucose (%) DP2 (%) DP3 (%) DP4+ (%) Ratio 1 Ratio 2 95.91 2.31 0.57 1.21 Ratio 10 96.71 2.15 0.60 0.54 Ratio 20 96.94 1.99 0.63 0.43 Ratio 60 97.07 1.90 0.63 0.40

[0053] As shown in Table 2, glucose was produced in high yield with the increasing total unit ratio of the diastatic enzyme to the isoamylase from 1:2 to 1:60. That is, the yield of glucose increased with the increasing ratio of the isoamylase to the diastatic enzyme. cl EXAMPLE 5

The Changes in Glucose Yield with the Varying Concentrations of Diastatic Enzyme in Multiple Reactions of the Debranching Enzyme Derived from an Acidothermus sp. and the Diastatic Enzyme

[0054] The changes in glucose yield with the varying concentrations of a diastatic enzyme (Glucoamylase, DuPont company) in multiple reactions of the debranching enzyme (isoamylase) and the diastatic enzyme were investigated. The enzymatic reaction conditions were as follows. Multiple enzymatic reactions with liquefied starch (DE 9.5) were performed at a temperature of 60 C. for 72 h, wherein the debranching enzyme and the diastatic enzyme were used in total unit ratios of 1:0.05, 1:0.04, 1:0.03, and 1:0.02, respectively. The reactions were quenched by heating at 100 C. for 5 min. Then, the amounts of glucose produced were measured from the ratios between the areas of peaks corresponding to the enzymatic reaction products by HPLC. As a result, the smaller the proportion of the diastatic enzyme, the higher the glucose yield. When the diastatic enzyme (glucoamylase) in the reaction for starch hydrolysis reached the equilibrium of hydrolysis, by-products (DP2) were formed. This problem was solved by lowering the concentration of the diastatic enzyme such that the formation of by-products is reduced, resulting in an increase in the relative yield of glucose.

TABLE-US-00004 TABLE 3 Debranching enzyme Diastatic enzyme (Isoamylase from (Glucoamylase, DuPont Acidothermus) company) Glucose (%) DP2 (%) DP3 (%) DP4+ (%) Ratio 1 Ratio 0.05 96.78 2.35 0.45 0.43 Ratio 0.04 96.90 2.08 0.53 0.49 Ratio 0.03 97.14 2.01 0.51 0.34 Ratio 0.02 96.25 2.05 1.22 0.48

[0055] As shown in Table 3, when the total unit ratio of the debranching enzyme to the diastatic enzyme changed from 1:0.05 to 1:0.03, the glucose yield increased. That is, the smaller the proportion of the diastatic enzyme, the higher the glucose yield.

[0056] COMPARATIVE EXAMPLE 1

The Comparison of Amylopectin Degradation Activities of Isoamylases Derived from Various Strains (Activity Evaluation under the Glycosylation Conditions (60 C., pH 4.3))

[0057]

TABLE-US-00005 TABLE 4 Activity No. Enzyme Strain (unit) 1 Isoamylase Enterobacter cloacae subsp. Cloacae 0 2 Bifidobacterium longum subsp. Longum 0 3 Thermotoga neapolitana 0 4 Escherichia coli str. K-12 substr. W3110 0 5 Sinorhizobium meliloti 0 6 Acidothermus cellulolyticus 130.18 7 Novosphingobium aromaticivorans 0 8 Thermotoga thermarum 0 9 Rhodothermus marinus 105.04

[0058] The amylopectin degradation activities of isoamylases derived from various strains under the glycosylation conditions were compared. The results are shown in Table 4. Only the enzymes derived from Acidothermus cellulolyticus and Rhodothermus marinus were found to be active for the degradation of amylopectin. Thereafter, the yields of glucose after treatment with the two enzymes were compared.

COMPARATIVE EXAMPLE 2

The Comparison of Glucose Yields after Treatment with the Debranching Enzymes (Isoamylases) Derived from Acidothermus cellulolyticus and Rhodothermus marinus

[0059] The yields of glucose from starch after treatment with the debranching enzymes derived from Acidothermus cellulolyticus and Rhodothermus marinus were investigated. The enzymatic reaction conditions were as follows.

[0060] First, Multiple enzymatic reactions with liquefied starch (DE 9.5) were performed at a temperature of 60 C. for 72 h, wherein a diastatic enzyme (Glucoamylase, DuPont company) and each of the debranching enzymes (isoamylase, pullulanase) were used in total unit ratios of 1:2 to 1:60. The reactions were quenched by heating at 100 C. for 5 min. Then, the amounts of glucose produced were measured from the ratios between the areas of peaks corresponding to the enzymatic reaction products by HPLC. As a result, higher glucose yields were obtained when the debranching enzyme (isoamylase) derived from Acidothermus cellulolyticus was used (see the results in Table 6) than when the debranching enzyme derived from Rhodothermus marinus was used (see the results in Table 5). In addition, starch sugars, including di-(DP2) and higher saccharides, were analyzed. As a result, when the debranching enzyme (isoamylase) derived from Acidothermus cellulolyticus was used, the proportions of the unreacted products (DP2, DP4+) which did not degrade to glucose were reduced, indicating relatively high yields of glucose.

TABLE-US-00006 TABLE 5 Diastatic enzyme Debranching enzyme (Glucoamylase, DuPont (Isoamylase derived from company) Rhodothermus) Glucose (%) DP2 (%) DP3 (%) DP4+ (%) Ratio 1 Ratio 2 95.88 2.48 0.35 1.29 Ratio 10 96.26 2.31 0.37 1.06 Ratio 20 96.34 2.24 0.44 0.98 Ratio 60 96.61 2.08 0.56 0.75

TABLE-US-00007 TABLE 6 Diastatic enzyme Debranching enzyme (Glucoamylase, DuPont (Isoamylase derived from company) Acidothermus) Glucose (%) DP2 (%) DP3 (%) DP4+ (%) Ratio 1 Ratio 2 95.91 2.31 0.57 1.21 Ratio 10 96.71 2.15 0.60 0.54 Ratio 20 96.94 1.99 0.63 0.43 Ratio 60 97.07 1.90 0.63 0.40

[0061] As shown in Tables 5 and 6, the higher glucose yields and smaller proportions of the unreacted products (DP2 and DP4) with relatively high degrees of polymerization were obtained when the isoamylase derived from Acidothermus cellulolyticus was used than when the debranching enzyme derived from Rhodothermus marinus was used. That is, the application of the inventive isoamylase to enzymatic reactions leads to an increase in glucose yield.